Pharmaceutical composition and method for regenerating myofibers in the treatment of muscle injuries

ABSTRACT

A pharmaceutical composition and method for regenerating cardiomyocytes in treating or repairing heart muscle damages or injuries caused by an ischemic disease. The pharmaceutical composition contains an active ingredient compound with a backbone structure of Formula (I). The active ingredient compound is capable of (a) increasing viability of myogenic precursor cells to enable said precursor cells to survive through an absolute ischemic period; (b) reconstituting a damaged blood supply network in said heart region where said injured muscle is located; and (c) enhancing cardiomyogenic differentiation efficiency of said precursor cells down cardiac linage, said steps being performed simultaneously or in any particular order.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/722,915, filed Jun. 27, 2007, which is a §371 national stageapplication of PCT/CN2006/002885, and claims priority to U.S.Provisional Application No. 60/791,462, filed Apr. 13, 2006, thecontents of which are hereby incorporated by reference. The applicationfurther claims priority to PCT Application Nos. PCT/IB2005/003202 andPCT/IB2005/003191, both filed Oct. 27, 2005, the contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a pharmaceutical composition and a method ofregenerating myocytes and myocardium for treating muscle damages.Particularly, it relates to a pharmaceutical composition and method forregenerating cardiomyocytes in treating or repairing heart muscledamages or injuries caused by an ischemic disease.

BACKGROUND OF THE INVENTION

Myocardial infarction (MI), or heart attack, is a disease due tointerruption of the blood supply to a part of the heart, causing damageor death of heart muscle cells. It is the leading cause of death forboth men and women over the world. Following myocardial infarction,there does not seem to be any natural occurring repairing processcapable of generating new cardiomyocytes to replace the lost musclecells. Instead, scar tissues may replace the necrosed myocardium,causing further deterioration in cardiac function.

Therapeutic replacement of the necrosed heart tissue with newlyregenerated functional cardiac myocytes is a treatment ideal that untilrecently has been unrealistic, because cardiac myocytes were consideredto be terminally differentiated, or in other words, the heart is apostmitotic nonregenerating organ. This dogma, however, has recentlybeen challenged by Beltrami et al, and others, who reported that apopulation of resident myocytes within the myocard ia can and doreplicate after infarction. In order to promote and improve the repairfor infarcted myocardia, transplantation of cardiomyocytes or skeletalmyoblasts has been attempted, but has not been very successful inreconstituting functional myocardia and coronary vessels.Transplantation of adult bone marrow-derived mesenchymal stem cells(MSCs) for cardiac repair following myocardial infarction has resultedin some angiogenesis and myogenesis, but the location of the newlyregenerated cardiac myocytes appeared mostly along the border zone wherethe blood supply is relatively less affected ¹⁻³.

Because acute myocardial infarction (MI) brings rapid damages or deathto myocytes (heart muscle cells), vascular structures and nonvascularcomponents in the supplied region of the ventricle, regeneration of newcardiac myocytes to replace the infarcted myocardia (heart musculartissues) in the central infarcted zone (the absolute ischemic region)through a sub-population of cardiac myocyte growth ⁴⁻⁸ ortransplantation of MSCs ¹⁻³ alone appears to be impossible without earlyreestablishment of the blood supply network locally. This probablyexplains why regeneration of cardiac myocytes following MSCstransplantation alone occurred mostly along the border zone adjacent tothe infarct where the blood supply is largely maintained ¹⁻¹¹.Therefore, the loss of myocardia, arterioles and capillaries in thecentral infarct area appeared to be irreversible, eventually leading toscar formation.

A more recent study ¹² reported that heart transplantation of MSCpre-modified with exogenous Akt in vitro produced a better result.Nonetheless, the regenerated cardiac myocytes could only infiltrate fromthe border zone into the scarred area, indicating that overexpression ofexogenous Akt, although enhancing the survival potential of thetransplanted MSCs, itself is insufficient to enable them to survive incentral ischemic regions. Furthermore, even in the less-ischemic borderzones, it was noted that the MSCs-derived regenerating cardiac myocyteswere scattered and seemed to have difficulty to cluster and formregenerating myocardia. This is probably due to poor cardiomyogenicdifferentiation efficiency of the survived transplanted MSCs. Theknowledge that natural cardiomyocyte reproduction, includingdifferentiation of residential progenitor myocytes or stem cellsrecruited from other sources, such as from endothelial cells or a nichein the bone marrow is insufficient to balance cardiomyocyte deathoccurred in acute or chronically damaged heart, has damped theenthusiasm of the researchers who thought myocardial regeneration wouldrepresent a promising method of treatment against heart diseases.

The prior art seems to teach that there are three major requirementscritical for regenerating functional myocytes in the entire areas ofinfarcted myocardia: 1) increased viability of the transplanted cells sothat they may survive through the absolute ischemic period, that is, theperiod from injection of the donor cells to formation of new vessels; 2)early reconstitution of the damaged blood supply network in theinfarcted myocardia to sustain the survival and efficient trafficking ofthe transplanted cells and maintain oxygenation and nutrient delivery;and 3) enhanced cardiomyogenic differentiation efficiency of thetransplanted cells to enable more survived donor cells to differentiatedown cardiac linage.

Therefore, to realize the therapeutic ideal of replacing necrosed hearttissues with newly regenerated functional cardiac myocytes, there is aneed for new therapeutic approaches, for example, an approach usingchemical compounds possessing biological properties that sufficientlysatisfy the aforementioned three requirements in order to serve thetherapeutic needs for treating myocardial infarction.

SUMMARY OF THE INVENTION

As one object of the present invention, there is provided apharmaceutical composition comprising a compound selected from the groupof chemical compounds sharing a common backbone structure of formula(I). The compounds have potent beneficial therapeutic effects not onlyon the survival potential and cardiogenic differentiation efficiency ofMSCs ex vivo, but also on repairing of MI in vivo. These compoundsthemselves are known in the art but they are never known as possessingthe above biological activities and therapeutic effects. They may beisolated from natural resources, particularly from plants or they mayalso be obtained though total or semi-chemical syntheses, with existingor future developed synthetic techniques. The backbone structure itselfpossesses the aforementioned myogenic effects and various variants canbe made from the backbone structure through substitution of one or morehydrogen atoms at various positions. These variants share the commonbackbone skeleton and the myogenic effects. Of course, they may vary inmyogenic potency.

The backbone structure of Formula (I) may have one or more substituentsattached. A substituent is an atom or group of atoms substituted inplace of the hydrogen atom. The substitution can be achieved by meansknown in the field of organic chemistry. For example, through a properdesign, high through-put combinatorial synthesis is capable of producinga large library of variants or derivatives with various substituentsattached to various positions of a backbone structure. The variants orderivatives of formula (I) may then be selected based on an activitytest on mesenchymal stem cells (MSCs), which can quickly determinewhether a particular variant could enhance proliferation and cardiogenicdifferentiation of the cultured MSCs. As used in this application, theterm “the compound of formula (I)” encompasses the backbone compounditself and its substituted variants with similar biological activities.Examples of these variants are presented in the following, all of whichpossess similar effects in terms of regenerating functional myocytes asthe backbone structure (i.e., the base compound itself):

Furthermore, as a therapeutic agent, the compound of formula (I) may bein a form of “functional derivatives” as defined below.

It is contemplated, as a person with ordinary skill in the art wouldcontemplate, that the above compounds may be made in various possibleracemic, enantiomeric or diastereoisomeric isomer forms, may form saltswith mineral and organic acids, and may also form derivatives such asN-oxides, prodrugs, bioisosteres. “Prodrug” means an inactive form ofthe compound due to the attachment of one or more specialized protectivegroups used in a transient manner to alter or to eliminate undesirableproperties in the parent molecule, which is metabolized or convertedinto the active compound inside the body (in vivo) once administered.“Bioisostere” means a compound resulting from the exchange of an atom orof a group of atoms with another, broadly similar, atom or group ofatoms. The objective of a bioisosteric replacement is to create a newcompound with similar biological properties to the parent compound. Thebioisosteric replacement may be physicochemically or topologicallybased. Making suitable prodrugs, bioisosteres, N-oxides,pharmaceutically acceptable salts or various isomers from a knowncompound (such as those disclosed in this specification) are within theordinary skill of the art. Therefore, the present invention contemplatesall suitable isomer forms, salts and derivatives of the above disclosedcompounds.

As used in this application, the term “functional derivative” means aprodrug, bioisostere, N-oxide, pharmaceutically acceptable salt orvarious isomer from the above-disclosed specific compound, which may beadvantageous in one or more aspects compared with the parent compound.Making functional derivatives may be laborious, but some of thetechnologies involved are well known in the art. Various high-throughputchemical synthetic methods are available. For example, combinatorialchemistry has resulted in the rapid expansion of compound libraries,which when coupled with various highly efficient bio-screeningtechnologies can lead to efficient discovering and isolating usefulfunctional derivatives.

The pharmaceutical composition of the present invention is useful fortreating myocardial injuries or necrosis caused by a disease,particularly MI, through regenerating heart tissues. The pharmaceuticalcomposition may be formulated by conventional means known to peopleskilled in the art into a suitable dosage form, such as tablet, capsule,injection, solution, suspension, powder, syrup, etc, and be administeredto a mammalian subject having myocardial injuries or necrosis. Theformulation techniques are not part of the present invention and thusare not limitations to the scope of the present invention.

The pharmaceutical composition of the present invention may beformulated in a way suitable for oral administration, systemicinjection, and direct local injection in the heart or implantation in abody part for long-term slow-releasing.

In another aspect, present invention provide a method for treating orameliorating a pathological condition in a mammal, where thepathological condition, as judged by people skilled in medicine, can bealleviated, treated or cured by regenerating functional cardiomyocytesand where the method comprises administering to the mammal with thepathological condition a therapeutically effective amount of a compoundof formula (I) and or its functional derivatives.

In another aspect, the present invention provides a method ofregenerating functional cardiomyocytes in a mammal who needs to replacedead or damaged heart tissues caused by a heart disease, such as,myocardial infarction (MI). This is a cell-transplantation basedtherapeutic approach, involving the steps of: (a) obtaining stem cells,such as MSCs; (b) contacting the stem cells with a compound of theformula (I) or their functional derivatives to activate the pathways ofcardiogenic differentiation prior to transplantation and (c) thentransplanting the activated cells into the infarcted heart tissues ofthe mammal. This therapeutic approach is capable of achieving thefollowing goals: 1) enhanced survival potential of the transplantedcells; 2) early reconstitution of blood supply network, and 3) enhancedcardiomyogenic differentiation efficiency of the transplanted cells byex vivo activation of MSCs forming cardiogenic progenitors prior totransplantation.

In another aspect, the present invention provides a method for treatingischemic heart diseases, particularly MI in mammals, which comprisingthe steps: (a) culturing MSCs or endothelial cells with a compound offormula (I) or their functional derivatives, (b) gathering theconditioned medium of the treated cells, which contains secretaryproteins that are active in driving heart infarction repair orcardiogenic differentiation of MSCs, and (c) administering or deliveringthe conditioned medium to the heart tissue in the infarct area.

In still another aspect, the present invention provides a researchreagent for scientific research on cardiogenic transdifferentiation ofstem cells, such as MSCs. The reagent comprises one or more compounds offormula (I) or their functional derivatives. It may be in a solid formor a liquid form. For example, it may be a solution of DMSO.

The various features of novelty which characterize the invention arepointed out with particularity in the claims annexed to and forming apart of this disclosure. For a better understanding of the invention,its operating advantages, and specific objects attained by its use,reference should be made to the drawings and the following descriptionin which there are illustrated and described preferred embodiments ofthe invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 outlines the process of isolating Niga-ichigoside Fl (referred toas “CMF”) from the plant of Geum Japonicuin as an example of making thecompound of the present invention.

FIG. 2 shows the effects of CMF on cardiogenic differentiation of theMSCs and up-regulation of phospho-Akt1 expression ex vivo.

FIG. 3 shows the therapeutic effect of a treatment based ontransplantation of CMF-pretreated MSCs.

FIG. 4 shows distribution ejection fraction (EF) and fractionalshortening (FS) after 2 days and 2 weeks cell transplantation in threegroups of rats (A: normal group; B: MI group transplanted withCMF-treated MSCs; C: MI group transplanted with MSCs not treated withCMF).

FIG. 5 shows the therapeutic effects of CMF on myocardial infarction(MI) animal model.

FIG. 6 shows the enhanced proliferation of cultured MSCs and myocardialregeneration induced by conditioned medium.

FIG. 7 shows the cellular source for CMF-induced myocardial regenerationin animal MI model.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS I. Experiment Procedures

All protocols used in the present invention conformed to the Guide forthe Care and Use of Laboratory Animals published by the U.S. NationalInstitutes of Health, and were approved by the Animal ExperimentalEthical Committee of The Chinese University of Hong Kong.

For the following discussion, CMF refers to the base compound (orbackbone compound) of the present invention. Its chemical structure isdefined by formula (I) shown in the above.

Obtaining Compounds of the Present Invention:

The compounds can be prepared from plants, although it may possible tomake it through chemical synthesis.

As an example for illustrating the process of preparing the compoundsfrom natural resources, the following provides details involved in CMF'sisolation and purification from one plant species, Geum Japonicum. Otherplants that may contain CMF or variants include, for example, Acaenapinnatifida R. et P., Agrimonia pilosa Ledeb, Asparagus filicinus,Ardisia japonica, Campsis grandiflora, Campylotropis hirtella (Franch.Schindl.), Caulis Sargentodoxae, Cedrela sinensis, Chaenomeles sinensisKOEHNE, Debregeasia salicifolia, Eriobotrya japonica calli, Eriobotryajaponica LINDL. (Rosaceae), Goreishi, Leucoseptrum stellipillum,Ludwigia octovalvis, Perilla frutescens, Perilla frutescens (L.) Britt.(Lamiaceae), Physocarpus intermedius Potentilla multifida L., Poteriumancistroides, Pourouma guianensis (Moraceae), Rhaponticum uniflorum,Rosa bella Rehd. et Wils., Rosa laevigata Michx, Rosa rugosa, Rubusalceaefolius Poir, Rubus allegheniensis, Rubus coreanus, Rubusimperialis, Rubus imperialis Chum. Schl. (Rosaceae), Rubus sieboldii,Rumex japonicus, Salvia trijuga Diels, Strasburgeria robusta, Strawberrycv. Houkouwase, Tiarella polyphylla, Vochysia pacifica Cuatrec,Zanthoxylum piperitum, etc.

Isolation of Cardiomyogenic factor (CMF) from Geum japonicum:

Referring to FIG. 1, the plant of Geum japonicum collected from GuizhouProvince of China in August was dried (10 kg) and percolated with 70%ethanol (100 L) at room temperature for 3 days twice. The extract wascombined and spray-dried to yield a solid residue (1 kg). The solidresidue was suspended in 10 liter H₂O and successively partitioned withchloroform (10 L) twice, then n-butanol (10 L) twice to produce thecorresponding fractions. The n-butanol (GJ-B) soluble fraction wasfiltered and dried by spray drying to yield a powder fraction, which wasconfirmed for their specific ability to stimulate cardiogenicdifferentiation of MSCs in cell culture in a way described below. It wasshown that n-butanol soluble fraction (GJ-B) could enhance theproliferation and cardiogenic differentiation of the cultured MSCs incell culture systems. The GJ-B fraction was then applied on a column ofSephadex LH-20 equilibrated with 10% methanol and eluted with increasingconcentration of methanol in water, resolving 7 fractions, GJ-B-1 toGJ-B-7. All the eluted fractions were tested for their activity with MSCculture systems. Activity test demonstrated that fraction 6 was mostactive in enhancing the cardiogenic differentiation of cultured MSCs.From GJ-B-6, a pure active compound was further isolated, which isreferred to as CMF through this disclosure. CMF's structure wasdetermined by NMR analysis and comparison with literature, and shown tobe of formula (I).

Preparation of MSCs for Transplantation:

The MSCs were cultured with CMF (10 μg/ml in growth medium) for 6 days.In parallel, the control MSCs were cultured in growth medium containingequivalent volume of 5% DMSO. On day 2, expression of endogenousphospho-Akt1 was assessed by immunocytochemistry and Western blot. Onday 4, myogenic differentiation was assessed by immunocytochemistry andWestern blot against MEF2, which were further confirmed byimmunocytochemistry and Western blot with an antibody specific to MHC onday 6. On day 3, both the CMF-pretreated MSCs and the control MSCs werelabeled with CM-DiI in culture and made ready for transplantation.

Preparation of Bone Marrow Mesenchymal Stein Cells:

The tibias/femur bones were removed from Sprague-Dawley (SD) rats andthe bone marrow (BM) was flushed out of the bones with IMDM culturemedium containing 10% heat inactivated FBS (GIBCO) and 1%penicillin/streptomycin. The BM was thoroughly mixed and centrifuged at1500 rpm for 5 minutes. The cell pellet was suspended in 5 ml growthmedium. The cell suspension was carefully put on 5 ml Ficoll solutionand centrifuged at 200 rpm for 30 min. The second layer, which containsBM cells was transferred into a tube and washed twice with PBS to removeFicoll (1200 rpm for 5 minutes). The cell pellet was resuspended in IMDMculture medium containing 10% heat inactivated FBS (GIBCO) and 1%penicillin/streptomycin antibiotic mixture. After 24 hours culture in a37° C. incubator with 5% CO₂, the non-adherent cells are discarded andthe adherent cells are cultured by changing medium once every 3 days andthe cells became nearly confluent after 14 days of culture. This was theBM cells, referred to as MSCs in the following, which were used for invitro and in vivo studies conducted in the present disclosure.

Western Blot Analysis:

Whole cell extracts of the CMF-treated cells or control cells wereprepared by lysing the cells with 3 times packed cell volume of lysisbuffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%Nonidet P-40, 10% glycerol, 200 mM NaF, 20 mM sodium pyrophosphate, 10mg/ml leupeptin, 10 mg/ml aprotinin, 200 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate) on ice for 30 minutes. Proteinyield was quantified by Bio-Rad DC protein assay kit (Bio-Rad). Equalamounts (30 μg) of total protein were size-fractionated by SDS-PAGE andtransferred to PVDM membranes (Millipore). The blots were blocked withphosphate-buffered saline plus 0.1% (vol/vol) Tween 20 (PBST) containing5% (wt/vol) milk powder (PBSTM) for 30 minutes at room temperature andprobed for 60 minutes with specific primary antibodies against ratphospho-Akt1 (mouse) or rat MHC (mouse, Sigma-Aldrich), diluted 1:1000in PBSTM. After washing extensively in PBST, the blots were probed byhorseradish peroxidase-coupled anti-mouse IgG (Amersham Biosciences) (1/1000 dilution in PBSTM, 60 min), extensively washed with PBST, anddeveloped by chemiluminescence.

Transplantation of the CMF Pretreated MSCs to the Heart Tissue:

The Sprague-Dawley (SD) rats were used and all animal procedures wereapproved by the University Animal Committee on Animal Welfare. Each ratwas anesthetized with intraperitoneal pentobarbital (50 mg/kg),intubated, and mechanically ventilated with room air using a Harvardventilator (model 683). After a left thoracotomy, myocardial infarctionwas induced by permanent ligation of left anterior descending (LAD)coronary artery. The 5×10⁵ DiI labeled CMF-pretreated MSCs (32 rats)suspended in saline were injected into three sites of the distalmyocardia (the ischemic region) of the ligated artery immediate afterthe ligation respectively (test group). The control rats were injectedwith an equivalent amount of DiI labeled non-treated control MSCs (32rats) suspended in saline at the same location and timing. For shamischemia (32 rats), thoracotomy was performed without LAD ligation.Sixteen rats subject to no-treatment were set as normal control.

Half of the experimental rats from different groups were sacrificedaccording to experimental plan on day 7 and day 14 post-infarction afterassessment of their heart function by echocardiography measurements. Thehearts of the sacrificed rats were removed, washed with PBS andphotographed respectively. All the specimens harvested were paraffinembedded and sectioned for tracing the signals of DiI and examination ofrevascularization, infarct size and regeneration of myocardia. If theregenerating cells were DiI positive, further MHC immonohistochemicalstaining was performed to confirm their cardiomyogenic differentiation.

Colocalization of the DiI label and cardiac-specific marker expressionwere examined with a confocal microscope (ZEISS, LSM 510 META). Briefly,the sections were immunohistochemically stained with rat-specifictroponin I antibodies. The confirmation of cardiomyogenicdifferentiation of the DiI labeled transplanted MSCs formingregenerating myocardia was carried out by merging the DiI-positivecells, indicating their donor cell origin, with the specific positivestaining of cardiac terminal differentiation marker-troponin I usingconfocal microscopic examination, implying their cadiomyogenicdifferentiation of these transplanted cells.

CMF Direct Treatment in MI Model:

Thirty-two SD rats were randomly divided into four groups: normal group,sham group, CMF-treated group and non-treated control group (8 ratseach). Rats were anesthetized with intraperitoneal pentobarbital (50mg/kg), intubated, and mechanically ventilated with room air using aHarvard ventilator (model 683). After a left thoracotomy, myocardialinfarction was induced by permanent ligation of left anterior descending(LAD) coronary artery. CMF in 5% DMSO (0.1 ml, containing 0.1 mg CMF)was injected into the distal myocardium (the ischemic region) of theligated artery in 8 rats immediate after the ligation (CMF-treatedgroup). Another 8 rats were injected with an equivalent amount of 5%DMSO at the same location and timing as non-treated control group. Forsham ischemia, thoracotomy was performed on 8 rats without LAD ligation.Further 8 rats without any treatment were set as normal group.

Conditioned Medium Containing Secretary Proteins from MSCs or OtherCells Induced by CMF:

The MSCs were treated with 10 ug/ml CMF for 24 hours toactivate/upregulate gene expressions and then washed thoroughly toremove residue of CMF. Then 5 ml of fresh growth medium was added to theculture and collected after another 3 days of culturing. The collectedmedium was referred to as conditioned medium. The 5 ml of conditionedmedium was condensed to a volume of 1 ml, and was used as a treatmentagent in the heart infarction animal model as described above. Briefly,after a left thoracotomy and ligation of LAD, 0.2 ml of the conditionedmedium was injected immediately into the distal part of ligation. Freshgrowth medium was used as control.

Bone Marrow Replacement with DiI Labeled MSCs:

Sixteen 5-week old SD rats were used for bone marrow transplantation.Recipient rats were irritated by 9.5 Gy of gamma irradiation from a137Cs source (Elite Grammacell 1000) at a dosage of 1.140 Gy/min tocompletely destroy the bone marrow derived stem cells of the rat.DiI-labeled MSCs (2×10⁸ cells suspended in 0.3 ml PBS) were theninjected through tail vein within 2 hours after irritation using a27-gauge needle. One week after irritation and transplantation, the ratswith DiI-labeled bone marrow were divided into two groups: one to betreated directly with CMF and the other as control without treatment.Heart infarction surgery and treatment scheme were performed asdescribed above. The experiment was terminated on day 14 post surgeryand treatment for further assessment. Heart specimens of the sacrificedrats were obtained. All the specimens were traced for DiI positive cellsand their cardiomyogenic differentiation by immunohistochemical stainingwith specific antibodies for heart type troponin I (Santa Cruz) and PCNA(Dako). Specific secondary antibody conjugated with alkaline phosphatase(Santa Cruz) was used to visualize the positively stained cells. DiIpositive signal was observed with a fluorescence microscope (Laica)

Estimation of Infarct Size:

Left ventricles from experimental rats sacrificed on day 14 were removedand sliced from apex to base in 3 transverse slices. The slices werefixed in formalin and embedded in paraffin. Sections (20 μm thickness)of the left ventricle were stained with Masson's trichrome, which labelscollagen blue and myocardium red. These sections were digitized and allblue staining was quantified morphometrically. The volume of infarct(mm³) of a particular section was calculated based on the thickness ofthe slice. The volumes of infarcted tissue for all sections were addedto yield the total volume of the infarct for each particular heart. Allstudies were performed by a blinded pathologist.

Angiogenic Assessment in Infarct Region:

Vascular density was determined on day 7 postinfarction from histologysections by counting the number of vessels within the infarct area usinga light microscope under high power field (HPF) (×400). Six random andnon-overlapping HPFs within the infarct filed were used for counting allnewly formed vessels in each section of all experimental hearts. Thenumber of vessels in each HPF is averaged and expressed as the number ofvessels per HPF.

Assessment of regenerating cardiac myocytes and myocardia: The sectionsfrom both CMF pretreated MSCs-transplanted and non-treatedMSCs-transplanted groups on day 7 post-ligation were stained with Ki67or myosin heavy chain (MHC) antibodies to identify the regeneratingmyocardia. Specific secondary antibody conjugated with alkalinephosphatase was used to visualize the positive stains. Briefly,paraffin-embedded sections were microwaved in a 0.1 M EDTA buffer andstained with a polyclonal rabbit antibody with specificity against ratKi67 at 1:3,000 dilution (Sant Cruz Biotechnology) and incubatedovernight at 4° C. After they were washed, the sections were incubatedwith a goat anti-rabbit IgG secondary antibody conjugated with alkalinephosphatase at 1:200 dilution (Sigma) for 30 min, and the positivenuclei were visualized as dark blue with a5-bromo-4-chloro-3-indolylphosphate-p-toluidine-nitro blue tetrazoliumsubstrate kit (Dako). The immediate neighbor sections from correspondingparaffin tissue block were incubated overnight at 4° C. in a 1:50dilution of rabbit anti-rat MHC (MF20, Developmental Hybridoma Bank,University of Iowa) antibodies, and further incubated for 30 minutes atroom temperature in a 1:100 dilution of peroxidase-conjugated goatanti-rabbit IgG (Sigma). After incubation with 1 mg/ml3,3′-diaminobenzidine (DAB; plus 0.02% H₂O₂), the slides wereinvestigated by microscopic analysis. The regenerating myocardium areawas delineated in the projected field by a grid containing 42 samplingpoints. Approximately, 30-60 calculating points along the border of aparticular regenerating myocardium were selected in each section. Thisgrid defined an uncompressed tissue area of 62,500 μm², which was usedto measure the selected 30-60 calculating points in each section. Theshapes and volumes of regenerating myocardia in the central area ofinfarct were determined by measuring in each section (50 μm apart) ofapproximately 70 sections the shapes and areas occupied by theregenerating myocardia and section thickness. Integration andcalculation with these variables produced a stereo-structure and yieldsthe volume of a particular regenerating myocardium in the central areaof the infarct in each section. Values and stereo structure of allsections of a particular tissue block were added and computed to obtainthe total volume and the full stereo-structure of the regeneratingmyocardia.

Echocardiography Assessment of Myocardial Function:

Echocardiographic studies were performed using a Sequoia C256 System(Siemens Medical) with a 15-MHz linear array transducer. The chest ofexperimental rats was shaved, the animal was situated in the supineposition on a warming pad, ECG limb electrodes were placed, andechocardiography was recorded under controlled anesthesia. Eachexperimental rat received a baseline echocardiography before theexperimental procedure. Two-dimensional guided M-mode andtwo-dimensional (2D) echocardiography images were recorded fromparasternal long- & short-axis views. Left ventricular (LV) end-systolicand end-diastolic dimensions, as well as systolic and diastolic wallthickness were measured from the M-mode tracings by using theleading-edge convention of the American Society of Echocardiography. LVend-diastolic (LVDA) and end-systolic (LVSA) areas were planimeteredfrom the parasternal long axis and LV end-diastolic and end-systolicvolumes (LVEDV and LVESV) were calculated by the M-mode method. LVejection fraction (LVEF) and fractional shortening (FS) were derivedfrom LV cross-sectional area in 2D short axis view:EF=[(LVEDV−LVESV)/LVEDV]×100% and FS=[(LVDA LVSA)/LVDA]×100%. Standardformulae were used for echocardiographic calculations. All data wereanalyzed offline with software resident on the ultrasound system at theend of the study. All measured and calculated indexes were presented asthe average of three to five consecutive measurements.

Statistics:

All morphometric data are collected blindly, and the code is broken atthe end of the experiment. Results are presented as mean±SD computedfrom the average measurements obtained from each heart. Statisticalsignificance for comparison between two measurements is determined usingthe unpaired two-tailed Student's t test. Values of P<0.05 areconsidered to be significant.

II. CMF-Induced Increased Survival Potential and CardiogenicDifferentiation of MSCs Ex Vivo

Referring to FIG. 2, following two days treatment with CMF (10 μg/ml) inthe culture, the expression of phospho-Akt1 was significantlyup-regulated compared with the untreated control as demonstrated byimmunocytochemical staining of the cells with antibodies specific tophospho-Akt1, positive cells being stained red mainly in the cytoplasm(FIG. 2 a: 1). Western blot confirmed that the increased expression ofphospho-Akt1 up to 3-4 folds over untreated cells (FIG. 2 b: 5A). Amongthe phospho-Akt1 up-regulated MSCs, more than 90% of them when culturedfor 2 additional days became positively stained with the antibodyspecific to myocyte enhancer factor 2 (MEF2), one of the earliestmarkers for the cardiogenic lineage, positive cells being stained orangein the nuclei (FIG. 2 a: 2) and confirmed by Western blot (FIG. 2 b:6A), indicating their commitment to cardiogenic differentiation. It wasnoted that the cultured MSCs were not all positively stained byanti-MEF2 antibody (FIG. 2 a: 2), as indicated by the blue nucleuspossibly because not all cultured MSCs were converted down thecardiogenic differentiation pathway by CMF or because some minorimpurities existed in the preparation of MSCs. Similarly, most of thecultured MSCs were positively stained with the antibody specific toheart type myosin heavy chain, positive cells being stained red in thecytoplasm (FIG. 2 a: 3) and confirmed by Western blot (FIG. 2 b: 7A)upon 6 days culture in the presence of CMF, while control cells werenegative stained by all three specific antibodies (FIGS. 2 a: 4 & 2 b:Bs). The sequential induction of MEF2 and MHC expressions confirmed theCMF-induced cardiogenic differentiation development of MSCs ex vivo. InFIG. 2 b, A stands for CMF-treated sample while B for untreated control.

III. Therapeutic Effect Via Transplantation of MSCs Pretreated with CMF

To determine whether the increased survival potential and cardiogenicdifferentiation efficiency showed ex vivo in MSCs treated with CMF priorto transplantation would bring about significant improvement inrepairing of MI in vivo, or in other words, whether CMF's ex vivoeffects have any therapeutic values, cell transplantation experimentswith MI animal model were performed, where MSCs pre-treated with CMFwere implanted in the areas of infarct. The homing, survival,proliferation, cardiomyogenic differentiation and maturation of thetransplanted cells were traced by the positive signals of eitherDiI-florescence and by immunohistochemical staining for Ki67 and MHC insections, which were from the hearts on day 7 and day 14 post infarctionand cell transplantation. As shown in FIG. 3, DiI positive cells on day7 (FIG. 3: 1) with the characteristic phenotype of a cardiac myocytewere observed in the whole area of the infarct in the test myocardiumgroup, indicating their donor cell origin and whole infarct zonedistribution. In control group (untreated with CMF), only scattered DiIsignals around the infarct border could be seen (FIG. 3: 2). Thecolocalization of DiI signals (red) and cardiac specific troponin Iexpression (green) was observed (FIG. 3: 3-5) in the whole infarct zoneby confocal microscopy. The merged image of DiI-positive (red) andcardiac specific marker troponin I expression (green) resulted inyellow-red-green overlapping colors in the same cells, thus confirmingthe in vivo cardiomyogenic differentiation and maturation of thetransplanted MSCs pretreated ex vivo with CMF. It was also noted that afew troponin I positive cells (green) were not DiI positive (FIGS. 3: 3& 5), probably because some regenerating myocytes were not derived fromthe transplanted DiI-labeled-MSCs. Similarly, a few DiI positive cellsshown in the light blue circles (FIGS. 3: 4 & 5), were negative introponin I immunostaining, indicating a small fraction of thetransplanted cells did not commit cardiogenic differentiation in vivo,or impurities contained in the preparation of MSCs.

Formation of new vessels could be detected as early as 12 hours aftertransplantation and many more newly formed vessels and capillariesfilled with blood cells were observed in the whole infarct areas in thetest group in 24 hours (before any regenerating cardiac myocytes couldbe seen) and in 7 days post infarction (FIG. 3: 1, yellow circles). Thedensity of the newly formed vessels in the infarct area of the CMFpretreated MSCs transplanted myocardia was on average 8±2 per high powerfield (40×) (HPF) on day 7. However, the new vessels were notDiI-positive, indicating that the cellular source of the vessels may notbe derived from the donor cells. It is contemplated that the donor MSCsmay be activated by CMF-pretreatment to stimulate and upregulateangiogenesis specific signaling pathways that induce the expression ofcertain angiogenic factors, which directly enhances the process of earlyrevascularization in infarcted myocardia. By contrast, approximately 3±2vessels per HPF were observed in the infarcted myocardia ofnon-pretreated MSCs transplantated controls on day 7 (FIG. 3: 2, yellowcircles).

As shown in FIG. 3, a large number of donor cell derived myocytes wereclustered and organized into myocardial-like tissue in the infarct area,which were positively stained by antibodies specific to MHC (FIG. 3: 6,blue circles) and Ki67 (FIG. 3: 7, blue circles), indicating that thesetransplanted CMF-pretreated MSCs retained the division ability andcommitted cardiomyogenic differentiation after transplantation in vivo.Under a high power field, these myocardial-like tissues showed thetypical morphology of myocardium, except the size was smaller thanundamaged existing myocytes (FIG. 3: 9, blue circles). These highlyorganized regenerating myocardium-like tissues occupied averagely 70±8%of the total infarct volume on day 7 and replaced the infarctedmyocardia by 80±8.5% on average on day 14 post-infarction in the testmyocardium group (FIG. 3: 8, R, regenerated cardiac myocytes; N,preexisting normal cardiac myocytes). This replacement of the infarctedheart tissue was accompanied by significant functional improvement, asdemonstrated in the echocardiography measurements (FIG. 4 & Table 1). Incomparison with the non-pretreated MSCs transplanted MI group on day 2and 14 post infarction, ejection fraction (EF) of thepretreated-MSCs-transplanted MI hearts was significantly higher(59.79±2.33 vs 52.1±2.54, P=0.03) on day 2, and markedly increased(67.13±2.53 vs 53.3±2.31, P=0.001) on day 14. Similarly, fractionshortening (FS) of the transplanted MI heart were significantly higher(29.43±1.35 vs 24.07±1.47, P=0.01) on day 2 and was significantlyincreased (31.72±2.57 vs 23.49±1.99, P=0.002) on day 14. The significantimprovements in EF and FS are a solid reflection of the functionalrecovery of the cardiac myocytes (Table 1).

TABLE 1 The distribution of ejection fraction (EF) and fractionalshortening (FS). (mean ± _SE): EF (%) FS (%) 2 days§ 14 days 

2 days§ 14 days 

Normal (16) 71.03 ± 4.05 68.24 ± 4.79 35.65 ± 3.99 34.02 ± 3.27 Sham(32) 70.45 ± 2.67 71.34 ± 2.77 36.03 ± 2.76 35.86 ± 2.13 CMF-pretreated(32) 59.79 ± 2.33* 67.13 ± 2.53* 29.43 ± 1.35* 31.72 ± 2.57* MSC control(32)  52.1 ± 2.54  53.3 ± 2.31 24.07 ± 1.47 23.49 ± 1.99 §Sixteen ratsfor normal group and 32 rats for sham operated, CMF-pretreated and MSCcontrol groups respectively.

 , Eight rats for normal group and 16 rats for sham operated,CMF-pretreated and MSC control groups respectively. *EF, P = 0.03 on day2; P = 0.001 on day 14, and FS, P = 0.01 on day 2 and P = 0.002 on day14.

IV. Direct Therapeutic Effects in MI Model without Pre-Treating MSCs andTransplantation

Referring to FIG. 5, following direct local injection of CMF in MImodel, it was found two weeks post infarction that in the control group(without CMF treatment) the myocardium on the distal part of theligation site became substantially white on visual inspection due toischemic necrosis (FIG. 5: 2). By contrast, the equivalent part inCMF-treated hearts were relatively red in appearance probably due toneovascularization (FIG. 5: 1), which was comparable to the non-ischemicparts of the heart and the sizes of infarct were significantly smallerthan those in the control hearts (FIG. 5: 2). Moreover, on transect ofthe infarct area, the left ventricle walls of the CMF treated hearts(FIG. 5: 3) were significantly thicker than those in control hearts(FIG. 5: 4). Histological observations revealed that the infarct sizesin CMF-treated hearts (n=8) were on average approximately ⅓-½ timessmaller than those in the control hearts (n=8), as was calculated bymeasuring the infarct volume in the left ventricular free wall on day 14after ligation. By Masson's Trichrome staining, it was found that in CMFtreated hearts, myocyte-like cell clusters, which were arranged inalmost the same orientation as the infarcted myocardium or theneighboring viable myocardia, were distributed in most parts of thewhole infarct regions (FIG. 5: 5). By contrast, the infarcted regions incontrol hearts were almost completely occupied by fibrous tissuereplacement, leaving almost no space for any possible cardiomyocytesregeneration, if any (FIG. 5: 6). Under a higher power field, in a sharpcontrast to the overall blue stained fibrous scar in control group (FIG.5: 8), the whole infarct areas in the CMF treated group were filled withregenerating myocyte clusters, well shaped to bear myocardial morphologywith little fibrous tissue in between (FIG. 5: 7), although the sizes ofthese regenerating myocytes were smaller than the neighboringpreexisting myocytes, probably because they were still on the way ofmaturing.

Furthermore, echocardiography demonstrated that the replacement ofinfarcted heart tissue with structurally integrated regeneratingmyocardia and reconstituted vasculatures was accompanied by significantfunctional improvement by day 2 post-infarct in CMF-treated hearts, andfurther improvement by day 14 compared with control hearts, probably dueto the growth and maturation of the regenerated myocardia andvasculatures that repaired the infarct.

Therapeutic Effect Via Conditioned Medium Induced by CMF-Treated MSCs

To determine whether conditioned medium containing certain inducedproteins secreted by CMF-activated-MSCs would bring about similareffects as CMF direct application or transplantation of theCMF-pretreated-MSCs to the infarct area, the conditioned medium wastested with both MSCs culture and heart infarction animal model.Referring to FIG. 6 a, after treatment with conditioned medium for 24hrs, the proliferation rate of MSCs increased to 120% compared tocontrol medium (fresh growth medium). Referring to FIG. 6 b, localinjection of conditioned medium to the ischemic region of MI model,cardiomyocyte regeneration was observed. Briefly, many regeneratingcardiomyocytes and many newly formed vessels filled with blood cellswere observed in the whole infarct zone (FIG. 6 b: 2) compared with thefibrous replacement in control (FIG. 6 b: 1).

V. Cellular Origin of the Regenerated Myocardia after Direct CMFTreatment

Referring to FIG. 7, studies on MI model with bone marrow replacementwith DiI labeled MSCs have provided direct evidence that the cellularorigin of the regenerating myocardium were derived from bone marrowMSCs. One week after bone marrow replacement, heart infarction surgerywas performed as described in the above. Fourteen days post infarction,it was found that the whole infarct regions in CMF-treated hearts werewell occupied by DiI labeled cells, which were largely absent in anynon-infarcted regions of having the preexisting viable cardiomyocytes.These DiI positive cells were clustered together bearing myocardial-likemorphology, but smaller in size compared with pre-existingcardiomyocytes (FIG. 7: 1). By contrast, only a few scattered DiIpositive cells were observed along the infarct border zone in controlinfarcted hearts (FIG. 7: 2). To confirm that these well organized DiIpositive cells were regenerating myocardia, immunohistochemistry withantibodies specific to troponin I and PCNA were performed. It was foundthat numerous DiI positive cells distributed in the whole infarct zonewere positively stained by specific antibodies for troponin I (FIG. 7:3) or PCNA (FIG. 7: 4). These DiI and troponin I or PCNA positivelystained cells were organized into myocardial-like tissue in the wholeinfarct zone in CMF treated hearts (FIGS. 7: 3 & 4). Under higher powerfield, these regenerating myocardial-like tissues showed the typicalmorphology of myocardium with clear intercalated disk connection betweenregenerating myocytes, indicating the ultrastructural maturation of theindividual regenerating myocyte into integrated myocardium (FIG. 7: 5).Without the structural integration between the regenerating myocytes,and between the regenerating myocardium and pre-existing viablemyocardium, functional integration and synchronous mechanical activitywould not be guaranteed. These regenerating myocardia occupied averagely69.3% of the total infarct volume on day 14 post-infarct. By contrast,in 6 control hearts, only a few cells were both troponin I and DiIpositive and were scattered around the vessels while the infarction zonewas mainly occupied by fibrous scar (FIG. 7: 6). These resultsdemonstrated that the CMF induced regenerating myocardium was functionaland derived from bone marrow MSCs.

IV Manufacturing Pharmaceutical Compositions and their Uses in TreatingIschemic Heart Diseases in Mammals

Once the effective chemical compound is identified and partially orsubstantially pure preparations of the compound are obtained either byisolating the compound from natural resources such as plants or bychemical synthesis, various pharmaceutical compositions or formulationscan be fabricated from partially or substantially pure compound usingexisting processes or future developed processes in the industry.Specific processes of making pharmaceutical formulations and dosageforms (including, but not limited to, tablet, capsule, injection, syrup)from chemical compounds are not part of the invention and people ofordinary skill in the art of the pharmaceutical industry are capable ofapplying one or more processes established in the industry to thepractice of the present invention. Alternatively, people of ordinaryskill in the art may modify the existing conventional processes tobetter suit the compounds of the present invention. For example, thepatent or patent application databases provided at USPTO officialwebsite contain rich resources concerning making pharmaceuticalformulations and products from effective chemical compounds. Anotheruseful source of information is Handbook of Pharmaceutical ManufacturingFormulations, edited by Sarfaraz K. Niazi and sold by Culinary &Hospitality Industry Publications Services.

As used in the instant specification and claims, the term “plantextract” means a mixture of natural occurring compounds obtained fromparts of a plant, where at least 10% of the total dried mass isunidentified compounds. In other words, a plant extract does notencompass an identified compound substantially purified from the plant.The term “pharmaceutical excipient” means an ingredient contained in adrug formulation that is not a medicinally active constituent. The term“an effective amount” refers to the amount that is sufficient to elicita therapeutic effect on the treated subject. Effective amount will vary,as recognized by those skilled in the art, depending on the types ofdiseases treated, route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatment. A personskilled in the art may determine an effective amount under a particularsituation.

While there have been described and pointed out fundamental novelfeatures of the invention as applied to a preferred embodiment thereof,it will be understood that various omissions and substitutions andchanges, in the form and details of the embodiments illustrated, may bemade by those skilled in the art without departing from the spirit ofthe invention. The invention is not limited by the embodiments describedabove which are presented as examples only but can be modified invarious ways within the scope of protection defined by the appendedpatent claims.

REFERENCES

-   1. Orlic, D. et al. Bone marrow cells regenerate infarcted    myocardium. Nature 410, 701-5 (2001).-   2. Toma, C., Pittenger, M. F., Cahill, K. S., Byrne, B. J. &    Kessler, P. D. Human mesenchymal stem cells differentiate to a    cardiomyocyte phenotype in the adult murine heart. Circulation 105,    93-8 (2002).-   3. Tomita, S. et al. Autologous transplantation of bone marrow cells    improves damaged heart function. Circulation 100, 11247-56 (1999).-   4. Beltrami, A. P. et al. Evidence that human cardiac myocytes    divide after myocardial infarction. N Engl J Med 344, 1750-7 (2001).-   5. Kajstura, J. et al. Myocyte proliferation in end-stage cardiac    failure in humans. Proc Natl Acad Sci USA 95, 8801-5 (1998).-   6. Laugwitz, K. L. et al. Postnatal isl1+cardioblasts enter fully    differentiated cardiomyocyte lineages. Nature 433, 647-53 (2005).-   7. Leferovich, J. M. et al. Heart regeneration in adult MRL mice.    Proc Natl Acad Sci USA 98, 9830-5 (2001).-   8. Poss, K. D., Wilson, L. G. & Keating, M. T. Heart regeneration in    zebrafish. Science 298, 2188-90 (2002).-   9. Reinecke, H., Zhang, M., Bartosek, T. & Murry, C. E. Survival,    integration, and differentiation of cardiomyocyte grafts: a study in    normal and injured rat hearts. Circulation 100, 193-202 (1999).-   10. Taylor, D. A. et al. Regenerating functional myocardium:    improved performance after skeletal myoblast transplantation. Nat    Med 4, 929-33 (1998).-   11. Yoo, K. J. et al. Heart cell transplantation improves heart    function in dilated cardiomyopathic hamsters. Circulation 102,    111204-9 (2000).-   12. Mangi, A. A. et al. Mesenchymal stem cells modified with Akt    prevent remodeling and restore performance of infarcted hearts. Nat    Med 9, 1195-201 (2003).

What is claimed is:
 1. A method of regenerating myocytes or myocardia inthe heart of a mammalian subject suffering an injured heart muscle,comprising a step of administering an effective amount of a compoundwith a backbone structure showing in formula (I) or a functionalderivative of said compound.


2. The method of claim 1, wherein said compound is said backbonestructure itself without any substitution.
 3. The method of claim 1,wherein said compound is selected from the group consisting of:


4. The method of claim 3, wherein said compound is:


5. The method of claim 4, wherein said injured heart muscle is caused byan ischemic event.
 6. The method of claim 5, wherein said ischemic eventis myocardial infarction.
 7. The method of claim 1, wherein saidmyocytes or myocardia are regenerated in a process comprising one ormore steps of (a) increasing viability of myogenic precursor cells toenable said precursor cells to survive through an absolute ischemicperiod; (b) reconstituting a damaged blood supply network in said heartregion where said injured muscle is located; and (c) enhancingcardiomyogenic differentiation efficiency of said precursor cells downcardiac linage, said steps being performed simultaneously or in anyparticular order.
 8. The method of claim 7, wherein said myogenicprecursor cells are mesenchymal stem cells coming from bone marrowthrough blood circulation.
 9. The method of claim 1, further comprisingthe steps of: (a) obtaining a plurality of stem cells; (b) contactingsaid stem cells with said compound or said functional derivative for aperiod of time; and (c) transplanting said cells into an infarcted ordamaged heart tissue of said mammalian subject.
 10. The method of claim1, further comprising the steps of: (a) formulating said compound orsaid functional derivative into a dosage form and (b) systematicallyadministering said compound or said functional derivative in said dosageform to said mammalian subject.
 11. The method of claim 10, wherein saiddosage form is selected from the group consisting of tablet, capsule,injection solution, syrup, suspension and powder.
 12. The method ofclaim 1, further comprising the steps of: (a) culturing a plurality ofMSCs or endothelial cells in a culture medium containing said compoundor said functional derivative for a period of time; (b) collecting saidculture medium, containing secretary proteins from said MSCs orendothelial cells; and (c) administering or delivering said medium toheart tissues in a infarct area.
 13. The method of claim 1, wherein atleast 95% by weight of said composition is identified compounds.
 14. Themethod of claim 1, further comprising adding a piece of information onusefulness of said compound, wherein said information indicates thatsaid compound is beneficial to a human suffering or having suffered aheart disease.
 15. The method of claim 1, further comprising adding apiece of information on usefulness of said compound, wherein saidinformation indicates that said compound is beneficial to regeneratemyocytes or myocardia in the heart of said mammalian subject.